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Simultaneous quantification of major bioactive constituents from Zhuyeqing Liquor by HPLC-PDA

Abstract

Background

Zhuyeqing Liquor (ZYQL) is a famous traditional Chinese functional liquor. For quality control of ZYQL products, quantitative analysis using high-performance liquid chromatography coupled with photodiode array detector (HPLC-PDA) was undertaken.

Methods

Eighteen compounds from ZYQL were simultaneously detected and used as chemical markers in the quantitative analysis, including 3-hydroxy-4,5(R)-dimethyl-2(5H)-furanone (M1), isobiflorin (M2), vanillic acid (M3), biflorin (M4), genipin 1-O-β-d-gentiobioside (M5), 1-sinapoyl-β-d-glucopyranoside (M6), geniposide (M7), epijasmnoside A (M8), ferulic acid (M9), luteolin 8-C-β-glucopyranoside (M10), isoorientin (M11), narirutin (M12), hesperidin (M13), 6′-O-sinapoylgeniposide (M14), 3,5-dihydroxy-3′,4′,7,8-tetramethoxyl flavones (M15), 3′,4′,3,5,6,8-hexamethoxyl flavone (M16), kaempferide (M17), and tangeretin (M18).

Results

The separation by gradient elution was achieved on SHIMADZU VP-ODS column (4.6 × 150 mm, 5 μm) at 30°C with methanol (A)/0.1% phosphoric acid (B) as the mobile phase. The detection wavelengths were 254, 278, and 335 nm. The optimized HPLC method provided a good linear relation (r ≥ 0.9991 for all the target compounds), satisfactory precision (RSD values less than 1.47%) and good recovery (97.40% to 103.44%). The limits of detection ranged between 0.20 × 10−4 and 64.90 × 10−4 μg/μL for the different analytes. Furthermore, the optimum sample preparation was obtained from HPD100 column eluted with water and 95% ethanol, respectively.

Conclusions

Quality control of ZYQL products, in total seven samples and twelve parent plants, was examined by this method, and results confirmed its feasibility and reliability in practice.

Background

Zhuyeqing Liquor (ZYQL), authorized as a functional health liquor in 1998 by the Ministry of Public Health in China, is a famous traditional Chinese functional liquor. The history of ZYQL could be traced back to the Warring States Period and became popular among people in the South and North Dynasties. In the Tang Dyansty and Song Dynasty, it had reached its climax (Yang [2007]). ZYQL was designed based on the principles of traditional Chinese medicine (TCM) and comprises 12 herbs: Lophatherum gracile Brongn. (Zhuye), Gardenia jasminoides Ellis (Zhizi), Lysimachia capillipes Hemsl. (Paicao), Angelica sinensis (Oliv.) Diels (Danggui), Kaempferia galanga L. (Shannai), Citrus reticulata Blanco (Chenpi), Chrysanthemum morifolium Ramat. (Juhua), Amomum villosum Lour. (Sharen), Santalum album L. (Tanxiang), Eugenia caryophyllata Thunb. (Gongdingxiang), Aucklandia lappa Decne. (Guangmuxiang), and Lysimachia foenum-graecum Hance (Linglingxiang). According to its long-term history use, ZYQL has various biological properties such as anti-oxidant, anti-fatigue, and immunoenhancement (Han [2007]).

Up to now, many studies show solicitude for the color, smell, and taste of the health functional liquor; few studies pay close attention to its chemical constituents and quality control. Currently, chemical analytical methods for the quality control of ZYQL have not been established. Therefore, it is necessary to establish a rapid and effective method for the quantitative analysis of the health functional liquor. In this study, the system of high-performance liquid chromatography coupled with photodiode array detector (HPLC-PDA) was used for analyzing the chemical profile of ZYQL. This method includes many advantages like high speed detection, excellent peak shapes, less solvent usage, well-defined chemical constituents, and simultaneous detection of multi-constituents, which is better than fingerprinting. Thus, simultaneous determination by RP-HPLC method is suitable for quantitative analysis and can be used as an effective tool to evaluate herbal medicine products.

Methods

Chemicals and materials

Methanol (HPLC-grade) was purchased from Fisher Scientific Co. (Franklin, MA, USA). Water for HPLC analysis was purified by a Milli-Q water purification system (Millipore, Billerica, MA, USA). Phosphoric acid (analytical grade) was purchased from Tianjin Guangfu Chemical Reagent Co. Ltd. (Tianjin, China). Other solvents from Tianjin Guangfu Chemical Reagent Co. Ltd. (Tianjin, China) were all of analytical grade.

Reference compounds of M1 to M18 (Figure 1) were isolated previously from ZYQL by author, structures of which were elucidated by comparison of spectral data (UV, MS, 1H NMR, and 13C NMR) with the literature data (Lin et al. [2006]; Okamurα et al. [1998]; Huang et al. [2012]; Zhang and Chen [1997]; Ma et al. [2009]; Miyake et al. [2007]; Liu et al. [2011]; Chen et al. [2008]; Rayyan et al. [2005]; Kumarasamy et al. [2004]; Ke et al. [1999]; Yoo et al. [2002]; Dinda et al. [2011]; Esteban et al. [1986]; Ballester et al. [2013]; Wang et al. [2010]; Hòrie et al. [1998]). The purity of each reference standard was determined to be above 98% by HPLC analysis based on a peak area normalization method, detected by HPLC-PDA and confirmed by HR-ESI-TOF-MS and NMR spectroscopy.

Figure 1
figure 1

Structures of compounds M1 to M18.

The samples of different batch and different alcoholicity of ZYQL and the 12 parent plants were provided by Shanxi XinghuaCun Fen Jiu Group Co., Ltd. (Shanxi, China). The 12 parent plants were identified by Professor Jincai Lu (Shenyang Pharmaceutical University, Shenyang, China). The voucher specimen was deposited at Shenyang Pharmaceutical University (Shenyang, China) and registered under the number ZYQL 2011050101.

Instrumentation and chromatographic conditions

Chromatographic analysis was performed on Waters 2695 Alliance HPLC system (Waters Co., Milford, MA, USA) with Waters 2998 PDA detector. Chromatographic separation was carried on a SHIMADZU VP-ODS column (4.6 mm × 150 mm, 5 μm; Shimadzu, Kyoto, Japan) at a column temperature of 30°C using methanol (A) and 0.1% phosphoric acid (B) as mobile phase with the gradient elution procedure show in Table 1. The flow rate was set at 1.0 ml/min and the detection wavelengths were 254 nm (for compounds M1 to M5, M7, M8, and M17), 278 nm (for compounds M12 and M13), and 335 nm (for compounds M6, M9 to M11, M14 to M16, and M18), which were chosen based on the maximum absorption of all the tested compounds. The injection volume was 10 μL, and the analytes were well separated in chromatographic conditions above.

Table 1 Time program of the gradient elution

Standard solution preparation

Individual stock solutions were prepared by dissolving the standards in methanol to obtain 3-hydroxy-4,5(R)-dimethyl-2(5H)-furanone (M1) 19.920 mg mL−1, isobiflorin (M2) 8.330 mg mL−1, vanillic acid (M3) 5.802 mg mL−1, biflorin (M4) 3.911 mg mL−1, genipin 1-O-β-d-gentiobioside (M5) 4.405 mg mL−1, 1-sinapoyl-β-d-glucopyranoside (M6) 1.115 mg mL−1, geniposide (M7) 23.804 mg mL−1, epijasmnoside A (M8) 12.060 mg mL−1, ferulic acid (M9) 2.515 mg mL−1, luteolin 8-C-β-glucopyranoside (M10) 1.510 mg mL−1, isoorientin (M11) 2.203 mg mL−1, nairutin (M12) 1.032 mg mL−1, hesperidin (M13) 4.801 mg mL−1, 6′-O-sinapoylgeniposide (M14) 5.312 mg mL−1, 3,5-dihydroxy-3′,4′,7,8-tetramethoxyl flavones (M15) 5.021 mg mL−1, 3′,4′,3,5,6,8-hexamethoxyl flavone (M16) 15.005 mg mL−1, kaempferide (M17) 6.408 mg mL−1, and tangeretin (M18) 17.155 mg mL−1. A mixed solution containing all the 18 standards was prepared as accurately as 108 μL M1, 6.8 μL M2, 2.4 μL M3, 8.0 μL M4, 165 μL M5, 96 μL M6, 106 μL M7, 3.4 μL M8, 8.2 μL M9, 9.5 μL M10, 7.9 μL M11, 35 μL M12, 40 μL M13, 80 μL M14, 8.2 μL M15, 11 μL M16, 12 μL M17, and 4.6 μL M18 and were placed in a 2-mL flask with stopper, diluted with methanol to make sure the volume reached 2 mL. All prepared solutions were respectively stored in a refrigerator at 4°C when not in use.

Treatment for samples

For the analysis, 40 mL of ZYQL were evaporated in vacuum at 50°C to dryness. The dry residue was processed as follows in order to obtain better analytical results: The residue was dissolved with water (10 mL) and applied to an HPD100 column eluted with water (150 mL); the water eluent was discarded and then eluted with 95% ethanol (150 mL). The 95% ethanol eluent was condensed and dissolved with methanol and then placed in a 2-mL flask with stopper, with a methanol-metered volume. Prior to HPLC analysis, the sample solution was passed through a 0.22-μm millipore filter.

The 12 crude dried parent plants were pulverized and sifted through 40 mesh sieve, respectively. One gram of the powder from the parent plant was placed in a 50-mL flask with stopper, then weighed again correctly, and extracted by ultrasonic method with 20 mL methanol for 30 min. Then standing, it was cooled down to room temperature (22°C) and the weight was mended to the incipient weight with methanol. Prior to HPLC analysis, the sample solution was passed through a 0.22-μm millipore filter.

Validation of the method

Calibration curves

Linearity was established by the injection of 1, 2, 4, 8, 12, 16, and 20 μL of the mixed reference standard solution prepared, respectively. Calibration graphs were plotted subsequently based on linear regression analysis of the integrated peak (Y) versus content (X, μg).

Limits of detection and quantitation

In order to evaluate the limits of detection (LODs) and the limits of quantification (LOQs) of the compounds, mixed standard stock solution was further diluted serially to provide a series of appropriate concentrations, and an aliquot of the diluted solutions was injected into HPLC for analysis. The LOD and LOQ for each analyte was calculated with corresponding standard solution on the basis of a signal-to-noise ratio (S/N) of 3 and 10, respectively.

Precision and stability

The precision of the chromatographic system was validated by injecting 10 μL of the mixed reference solution six times during 1 day. Stability study was performed with sample solution in 48 h (the time points are 0, 5, 10, 15, 25, 35, and 48 h, respectively). Variations were expressed by relative standard deviations (RSD) of peak area.

Repeatability and recovery

The repeatability test was analyzed by injecting six independently prepared samples (45° ZYQL (20130207), the concentration, and prepared method as the ‘Treatment for samples’). The RSD value of concentration was adopted to evaluate repeatability. The recovery tests were studied by adding the proper amount of mixed-reference standard solution to the sample (45° ZYQL (20130207)), and then processed by the method described in the ‘Treatment for samples’ section to yield the final concentration. The experiment was repeated six times.

Results and discussion

Optimization of chromatographic conditions

To improve resolution and sensitivity of analysis but reduce analytical time, the following chromatographic conditions were optimized (Gao et al. [2013]), including different mobile phase compositions (methanol, acetonitrile, and aqueous phosphatic acid of different concentrations), column temperature, and wavelength: To inhibit ionization of the acidic ingredients in the ZYQL sample, phosphatic acid was added in mobile phase. Two mobile phase systems, methanol-phosphatic acid aqueous solution and acetonitrile-phosphatic acid aqueous solution, were examined, and then column temperatures at 25°C, 30°C, 40°C, and 50°C were compared. A sensitive wavelength was determined by PDA with reference compounds. Present researches indicated that better separation and results were obtained using a mobile phase of water and methanol rather than water and acetonitrile. Therefore, in this work, the optimum resolution was achieved using methanol (A) and 0.1% phosphatic acid (B) as mobile phase, with a column temperature of 30°C at different detection wavelengths, which were described in ‘Instrumentation and chromatographic conditions’ section, with gradient elution (Table 1). All 18 standard analytes could be eluted with baseline separation in 90 min. Representative chromatograms for the mixed reference standard and 18 standard compounds were shown in Figure 2A,B.

Figure 2
figure 2

Stack views. (A) Different detector-wavelength HPLC chromatograms of mixed reference standards (from up to down: 335, 278, 254 nm). Column: SHIMADZU VP-ODS column (4.6 mm × 150 mm, 5 μm), temperature of 30°C. (B) HPLC chromatograms of M1 to M18 and mixed reference standards (from up to down: 254 nm M1, M2, M3, M4, M5, M7, M8, M17, mixed reference standards; 278 nm M12, M13, mixed reference standards; 335 nm M6, M9, M10, M11, M14, M15, M16, M18, mixed reference standards).

Optimization of sample preparation

In order to eliminate the water-soluble constituents and obtain the liposoluble constituents, the optimization of sample preparation was performed using 45° ZYQL (20130207). Forty milliliters of ZYQL was evaporated in vacuum at 50°C to dryness. And the following five methods were choosen to select the best method for sample preparation. First, the dry residue was suspended with water (10 mL) and extracted with acetoacetate (10 mL). The acetoacetate extract was condensed and then methanol was used to meter the volume (2 mL). Second, the dry residue was suspended with water (10 mL) and extracted with n-butanol (10 mL) The n-butanol extract was condensed and then methanol was used to meter the volume (2 mL). Third, the dry residues was dissolved with 70% ethanol (20 mL) to precipitate the polysaccharide and then condensed the supernate, use methanol to metered volume (2 mL). Fourth, the dry residues was dissolved with water (10 mL) as fraction A, then the remanent residues was dissolved with methanol (10 mL) as fraction B. Fraction A was applied to an SPE column eluted with water (150 mL); the water eluent was discarded; fraction B was applied to the same SPE column eluted with methanol (150 mL); the methanol eluent was condensed and methanol was used to meter the volume (2 mL). Fifth, the dry residue was dissolved with water (10 mL) and applied to an HPD100 column eluted with water (150 mL). The water eluent was discarded and then eluted with 95% ethanol (150 mL). The 95% ethanol eluent was condensed and then methanol was used to meter the volume (2 mL). Comparing the analytical results of the target constituents, though the former three methods proved to be more simple than the other, they could not obtain all the tested constituents and some content too lower to accurately reflect the real content. So, these three methods were deserted. The fourth one although could obtain all the tested constituents but at a lower content. Therefore, the optimized condition was selected, the fifth one (Table 2, Figure 3).

Table 2 Optimization of the treatment method of Zhuyeqing Liquor (μg/mL)
Figure 3
figure 3

Stack views of 45° Zhuyeqing Liquor preparation method HPLC chromatograms (254 nm, from up to down: method 1, method 2, method 3, method 4 and method 5).

Validation of the method

The method was validated in terms of linearity, LOD and LOQ, precision, repeatability, stability, and recovery test. All calibration curves exhibited good linearity (r ≥ 0.9991) in a relatively wide linear range as shown in Table 3. For the quantified compounds, the LOD and LOQ were 0.20 × 10−4~64.90 × 10−4 μg/μL and 0.67 × 10−4~216.34 × 10−4 μg/μL, respectively (Table 3), which were calculated with corresponding standard solution on the basic of a signal-to-noise ratio (S/N) of 3 and 10, respectively. Table 4 showed the results of precision, stability, recovery and repeatability of the 18 analytes. It was indicated that the RSD of the precision variations were less than 1.47% for all 18 analytes. The RSD of repeatability was less than 1.96% for all the analysis, which proved that this assay had good reproducibility. Stability test results, with RSD less than 1.76%, indicated that the sample solution was stable at room temperature for at least 48 h. The mean recovery rates, which ranged from 97.40% to 103.44% with RSD values less than 3.30% for the analytes concerned, showed that the developed analytical method had good accuracy. All these values fall within acceptable limits, which indicates this HPLC method is reliable with significant repeatability, recovery rate, and precision. The results proved that HPLC is appropriate for analyzing and assessing the quality of ZYQL.

Table 3 Regression equations, correlation coefficients, and linear range for 18 analytes in Zhuyeqing Liquor
Table 4 Precision, stability, recovery, and repeatability data of 18 analytes in Zhuyeqing Liquor

Sample analysis

The HPLC analytical method described above was subsequently used to simultaneously quantify 18 compounds in seven commercial products and 12 parent plants supplied by Shanxi XinghuaCun Fen Wine Group Co., Ltd. (Shanxi, China). Generally, the 18 compounds were authenticated by comparison of their retention times and MS spectra with those of reference standards. The representative HPLC chromatograms of mixed standard solution and sample solutions are shown in Figure 4. The analytical results are summarized in Tables 5 and 6. According to the chromatographic results shown in Table 5, there was no any constituents to be detected in 45° FenJiu (solvent of ZYQL). Moreover, the concentration of compounds M1 to M18 in 45° ZYQL were higher than those in 42° and 38°, which showed that with the increase of alcoholicity, the content of bioactive constituents increased as well. In addition, there was no content difference between the successive 5 years of 45° ZYQL. This indicated that the quality of 45° ZYQL was stable for at least 5 years.

Figure 4
figure 4

Stack views of different detector-wavelength HPLC chromatograms. (From up to down: 45° Zhuyeqing Liquor, 45° FenJiu, mixed reference standards, blank solvent: methanol, respectively).

Table 5 Contents of 18 analytes in different batches and different alcoholicity of Zhuyeqing Liquor (μg/mL)
Table 6 Contents of 18 analytes in 12 parent plants (mg/g)

Table 6 showed the content of compounds in 12 parent plants, which exhibited that the major bioactive constituents were mainly from Gardenia jasminoides Ellis (Zhizi), Kaempferia galanga L. (Shannai), Citrus reticulata Blanco (Chenpi), and Lysimachia foenum-graecum Hance (Linglingxiang). And this result was greatly useful and helpful for the quality control and further formula optimization of the technical study of Zhuyeqing Liquor.

Conclusions

An HPLC-PDA method has been developed for the simultaneous determination of 18 major compounds extracted from ZYQL for the first time. The validation data indicated that this method is reliable and can be applied to determine the contents of the 18 compounds in different ZYQL products. This valuable information concerning the concentration of these bioactive constituents in ZYQL could be of great importance for the quality assessment and should therefore be useful for the guidance of development of the new health care products. Furthermore, this HPLC-PDA assay supplies a rapidness and effectiveness method for the simultaneous determination of multiple constituents in ZYQL.

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Acknowledgements

Grateful acknowledgement is made to the Shanxi XinghuaCun Fen Jiu Group Co., Ltd. (Shanxi Province, China) and National Key Technology R&D Program (2012BAI30B02) for financial support of this work. The authors acknowledge Waters Co. Ltd. for Cooperation Laboratory.

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Correspondence to Jin-hui Wang.

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Authors' contributions

HYG carried out the whole experiment, SYW participated in the sample preparation, HYG and JHW performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.

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Gao, Hy., Wang, Sy., Wang, Hy. et al. Simultaneous quantification of major bioactive constituents from Zhuyeqing Liquor by HPLC-PDA. J Anal Sci Technol 5, 34 (2014). https://doi.org/10.1186/s40543-014-0034-1

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